The past decade has shown great advances in the area of growing tissues and organs in vitro (Langer et al., 1993, "Tissue Engineering," Science 260:920-926). One such system for culturing three-dimensional tissues is described in U.S. Pat. No. 5,266,480 to Naughton et al. The culture system of Naughton et al. involves seeding stromal cells from a tissue of interest onto a porous substrate. As the stromal cells grow in this environment, they produce an extracellular matrix and deposit growth factors that contribute to the development of a three-dimensional tissue. This static cell culture milieu provides the necessary microenvironment for cell-cell and cell-matrix communication as well as an adequate diffusional environment for delivery of nutrients and removal of waste products. When the stromal tissue has grown and has developed into a three-dimensional tissue, it is ready for the seeding of the parenchymal cells of interest. The resulting system provides an "in vivo" environment for the full differentiation of the tissue. This system has been used to culture bone marrow tissue (Naughton et al., 1987, "Hematopoiesis on Nylon Mesh Templates. I. Long Term Culture of Rat Bone Marrow Cells," J. Med. 18:219-250; Naughton et al., 1989, "Modulation of Long-Term Bone Marrow Culture by Stromal Support Cells," Ann. NY Acad. Sci. 554:125-140); skin tissue (Landeen et al., 1992, "Characterization of Human Dermal Replacement," Wounds 4:167-175; Naughton et al., 1989, "A Physiological Skin Model for In Vitro Toxicity Studies," 183-189, Alternative Methods in Toxicology. In vitro Toxicology: New Directions Vol. 7, (A. M. Goldberg, ed.) Mary Ann Liebert Publishers, New York; Slivka et al., 1993, "Characterization, Barrier Function, and Drug Metabolism of an In Vitro Skin Model," J. Invest. Dermatol. 100:40-46; U.S. Pat. No. 5,266,480); and liver tissue (Naughton et al., 1991, "Long Term Liver Cell Cultures as Potential Substrates for Toxicity Assessment," 193-202, In Vitro Toxicoloqy: Mechanisms and New Technology (A. M. Goldberg, ed.) Mary Ann Liebert Publishers, New York).
While many methods and bioreactors have been developed to grow tissue masses for the purposes described above, these bioreactors do not adequately simulate in vitro the mechanisms by which nutrients and gases are delivered to tissue cells in vivo. Cells in living tissue are "polarized" with respect to diffusion gradients. Differential delivery of oxygen and nutrients, as occurs in vivo by means of the capillary system, controls the relative functions of tissue cells and perhaps their maturation as well. Thus, prior art bioreactors that do not simulate these in vivo delivery mechanisms cannot be used to culture a wide variety of three-dimensional tissues.
The tissue culturing system and bioreactors of the present invention improve on the prior art methods of culturing three-dimensional tissues by using diffusion gradients to deliver nutrients to, while simultaneously removing metabolic waste products from, the three-dimensional tissue culture. Such a diffusion-driven delivery mechanism enhances delivery of nutrients and removal of waste products and simulates in vitro the diffusional mechanisms whereby nutrients are delivered to mammalian cells in vitro, thereby optimizing the growth and differentiation of cell cultures grown in vitro. Thus, a wide variety of three-dimensional tissues having multifunctional cells can be cultured and sustained using the present invention.
Currently available bioreactor techniques for growing tissue masses in general include hollow fiber techniques, static maintenance reactor systems, fluidized bed reactors, and flat-bed, single-pass perfusion systems.
The most commonly used bioreactors involve hollow fibers. Hollow fiber reactors generally use numerous hollow fiber membranes of appropriate composition and porosity for the cells being cultured. They are often referred to as artificial capillary systems. (See, for example, U.S. Pat. No. 4,200,689 to Knazek et al.) Culture medium flows through the middle of the hollow fibers, and the cells are located on the outside of the fibers and in the spaces between the fibers. Nutrients flow through the hollow fibers to the cells. This type of bioreactor is not capable of growing thick tissue, as the cells only grow in the small interstitial spaces between the hollow fibers.
Hollow fibers are also disclosed in U.S. Pat. No. 5,081,035 to Halberstadt et al. In this system, cells are also grown in the interstitial spaces of an array of capillary tubes. Convective forces are used to maintain a constant nutrient gradient to all of the cells growing in the interstitial areas of the bioreactor. This method is also limited to growing cells in the small areas between the fibers, and cannot be used for a thick tissue.
Another hollow fiber device is described in U.S. Pat. No. 3,997,396 to Delente. In this system, cells are attached to the interstitial spaces of a hollow fiber bundle. An oxygen carrier is passed through the center of the fibers while the cells are incubated in a nutrient medium. As with the previously described hollow fiber devices, this method does not provide a thick cultured tissue and does not utilize osmotic pressure differentials to deliver nutrients to the cells from the nutrient medium.
Yet another hollow fiber device is described in WO 90/13639 to Tolbert et al. However, this system does not utilize osmotic pressure differentials to deliver nutrients to the cultured tissue mass.
A single-pass perfusion bioreactor system is described in Halberstadt et al., 1994, "The In Vitro Growth of a Three-Dimensional Human Dermal Replacement Using a Single-Pass Perfusion System," Biotechnology and Bioengineering 43:740-746. In this system a tissue is cultured on a mesh contained in a teflon bag. Media containing nutrients is pumped through the bag using a peristaltic pump. Nutrients in the media diffuse into the tissue and waste products secreted into the media are carried away. While this system has been used to culture relatively thick skin tissue, the method does not drive the delivery of nutrients by way of a diffusion or osmotic pressure gradient.
Generally, the prior art has not found a way to culture a wide variety of three-dimensional tissues. As discussed above, the prior art bioreactors do not simulate in vivo nutrient delivery mechanism, and therefore these bioreactors cannot be used to culture a wide variety of tissues.
A system to provide hepatic assist to patients awaiting transplants or with limited functioning livers has also long been sought. Unlike the kidney, the function of which is basically only to filter, or unlike the heart, the function of which is purely mechanical, the liver has functions that are complex and involve the removal, chemical conversion, and addition to the blood of a multitude of chemicals, or combinations of these functions. Past methods of providing an artificial liver have failed to provide a device that is as effective as a human liver, and which can be used by patients with a wide range of liver malfunctions. The various methods used in the past are described, for example, in Takahashi et al., September, 1991, Digestive Diseases and Sciences 36(9).
One popular method has been charcoal hemoperfusion. This method is used for the treatment of acute hepatic failure. Charcoal is used to remove large molecules of high molecular weight from a patient's blood. However, charcoal hemoperfusion acts only to filter the blood and does not replace other complex liver functions such as the chemical conversion of ammonia to urea or cytochrome P450 activity, which is the main detoxification activity of the liver. In addition, chemical components that would be added to the blood by a healthy liver are not supplied using this method.
Another popular method utilizes microporous membranes to filter plasma from whole blood. Toxins are removed from the separated plasma by multiadsorbents and the purified plasma is reinfused through a microfilter which prevents the passage of fine particles of adsorbent back into the blood. This method, like charcoal perfusion, fails to add chemical components to blood that would be supplied by a healthy liver.
An additional method attempted has been the extracorporeal perfusion of a mammalian liver. While theoretically this method can perform the plethora of complex functions of a healthy liver, the method is difficult to perform and relies on donor organs which are difficult to obtain.
Other therapeutic modalities in use include plasma exchange, a hybrid artificial liver and variations thereof. In the plasma exchange method, the plasma of the patient is exchanged for fresh-frozen plasma. This method presents the danger of removing some indispensable substance from the patient's blood, and requires large amounts of normal plasma, which can be expensive and is not a permanent solution to liver failure.
For a hybrid artificial liver, mammalian liver cells are used to perform the functions of the liver that conventional synthetic systems are incapable of performing, probably due to the lack of a complete understanding of the functions of the liver. Various types of liver cells have been used including liver slices or pieces, liver clusters, and isolated hepatocytes. Experiments with this method have been hampered by the fact that the hepatocytes lose metabolic activity in culture over a short period of time.
Other prior art artificial livers have typically used a cultured monolayer of hepatocytes seeded onto a membrane. One such method is described in U.S. Pat. No. 3,734,851 to Matsuma. Briefly, a stream of blood from a living animal is flowed over a semi-permeable membrane that is in contact with a confluent mono-layer of liver cells. A dialysate is flowed over another semi-permeable membrane that is adjacent to the first semi-permeable membrane and which is also in contact with the confluent mono layer of cells. Waste products carried by the blood pass through the first membrane, are acted upon by the cell layer, pass out through the second membrane and are taken up by the dialysate liquid and carried away. At the same time, desirable metabolic products are taken up by the blood stream. Although these methods have also been used with slices of liver tissue, the tissue layers used have been limited to thickness of between 20-100 .mu.m, (U.S. Pat. No. 3,734,851 to Matsuma), which severely limits the capacity of blood that can be cleansed using this method.
Some other prior art liver assist devices have used artificial capillaries comprising small tubes made of a semi-permeable membrane. Liver cells are seeded on the outside of the tubes, and the medium flows through the capillary-like tubes, which have thin walls (100 .mu.m). Collagen is most often used to aid in attachment of hepatocytes to the tubes. Again, these devices are limited in the amount of blood that can be efficiently cleansed due to the lack of liver cells available to act on the blood.
Hollow fibers are used in a perfusion device described in U.S. Pat. No. 5,043,260 to Jauregul, in which a porous membrane separates a perfusion compartment from a hepatocyte compartment. The membrane is provided by hollow fibers communicating with perfusion inlets and outlets of the device, with the hepatocytes attached to the outer surface of the fibers. There is also a second set of hollow fibers communicating with a waste inlet and waste outlet.
In PCT application WO 93/16171 to Barker et al., glass beads are utilized as a matrix instead of tubes or a membrane to entrap hepatocytes and allow perfusion.
In general, however, the prior art has not found a way to make the hepatocytes proliferate adequately for a sustainable amount of cells or to form cell-cell interactions similar to liver in vivo. It is believed that one problem with prior art systems is the lack of ability to sustain the multifunctional cells of the liver, whose various functions are dictated at least in part by the cellular architecture of the tissue mass and the relative spacial relationship to the nutrient supply. Accordingly, none of the disclosed devices enable the growth of thick tissue that could be used as an effective extracorporeal liver device.